Revertaid first strand cdna synthesis kit

Total RNA was isolated from cells (1 × 10⁶) by using the NucleoSpinR^® RNA Kit II (Macherey-Nagel GmbH, Düren, Germany) in accordance to the manufacturer’s instructions. cDNA was reverse transcribed from mRNA using the RevertAid™ First Strand cDNA Synthesis Kit (VWR International, Darmstadt, Germany) are referred to instruction manual. For PCR (total volume 25 µL per reaction) 1.25 U Taq Polymerase, 1× reaction buffer, 2 mM MgCl₂, 2 µM dNTPs (all reagents from VWR International, Darmstadt, Germany) and 100 µM primers (Life Technologies, Darmstadt, Germany) were used. The cycling conditions comprised of an initial denaturation step (5 min at 95 °C), 35 cycles of amplification (30 s 94 °C; 30 s appropriate annealing temperature; 30 s 72 °C) and final elongation (10 min 72 °C). PCR products were separated on a 1% TAE agarose gel. PCR bands were visualized by GelRed™ staining (VWR International GmbH, Darmstadt, Germany) and the GelDoc^TM EZ Imager System (Bio-Rad, Munich, Germany). Primer pairs used in this study are summarized in Table 1.

RNA extractions from hMDMs and hAMs were performed using an RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. RNA content and quality was quantified and assayed, respectively, using a Nanodrop (Thermo Fisher Scientific) and RNA reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (VWR). Catalogued pre-designed gene primer probes for IL1β, TNFα, IL10, NFκB, IL12a, IL18, PFKFB3, GAPDH, PKM2, G6PD, RPIA, CPT1A, FASN, GLS, IDO1, and 18S were purchased and real-time RT-qPCR was performed using Taqman Universal Master Mix (Applied Biosystems) on a QuantStudio 5 RT-qPCR System (Applied Biosystems). Relative quantitative data was obtained and analyzed utilizing the 2^–ΔΔCt method as previously described (27 (link)). Secreted protein levels of IL1β, IL10 (BioLegend ELISA Max Deluxe kits) and TNFα (Invitrogen ready-set-go kit) present in cell supernatants were quantified by ELISA, according to the manufacturer’s instructions.

RNA was isolated from 1×10⁶ cells by using the NucleoSpin^® RNA II Kit from Macherey-Nagel (Macherey-Nagel GmbH, Düren, Germany) in accordance to the manufacturers’ instructions. Reverse Transcription of RNA into cDNA was performed using the RevertAid^™ First Strand cDNA Synthesis Kit (VWR International, Darmstadt, Germany) as referred to the instruction manual. PCR was performed in a 25μl reaction mixture containing 1.25U Taq Polymerase, 1× reaction buffer, 2mM MgCl2, 200μM of each dNTP (all reagents were purchased from VWR International, Darmstadt, Germany) and 100μM primers (Life Technologies, Darmstadt, Germany). The cycling conditions comprised of an initial denaturation of 5min at 94°C and 30 cycles of 0.5min at 94°C, 0.5min at the appropriate annealing temperature and 0.5min at 72°C followed by a final elongation for 10min at 72°C. All used primer pairs concomitant with their specific annealing temperature and product length are summarized in Table 1. PCR products were separated on a 1% agarose gel. Bands were visualized by GelRed^™ staining (VWR International GmbH, Darmstadt, Germany) and the GelDoc^™ EZ Imager system (Bio-Rad, Munich, Germany).